High capacity composite depth filter media with low extractables

ABSTRACT

A depth filtration device for the clarification of biological fluids including a composite depth filter media having a nonwoven first layer integral with a second layer containing a polyacrylonitrile (PAN) fibers, a filter aid, and a wet-strength resin. The depth filter media exhibits increased binding capacity for soluble impurities such as DNA and host cell proteins from biological/cell culture feedstreams during secondary clarification and low-level impurity clearance of harvested cell culture fluids, such as those used for the manufacture of monoclonal antibodies. The depth filter media additionally exhibits significantly lower flushing requirements, resulting in lower levels of organic, inorganic and bioburden extractables released, high dirt holding capacities and good chemical and/or radiation resistance.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/720,102, filed Dec. 19, 2019, which is a continuation of U.S.application Ser. No. 14/890,774, filed Nov. 12, 2015, which is a USNational Stage application of International Application No.PCT/US2014/053729, filed Sep. 2, 2014, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 61/871,985, filedAug. 30, 2013, the disclosure of each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

In general, the present invention relates to high capacity depth filtermedia with reduced pre-use flushing requirements, and an increasedbinding capacity for host cell proteins and other soluble impuritiescontained in biological product-containing feedstreams. Moreparticularly, it relates to high capacity depth filtration devices usedin the clarification of cell culture/biological feedstreams, whichutilizes porous depth filter media which incorporates an inorganicfilter aid having a sufficient surface area and adsorptive properties toextract soluble impurities from said feedstreams, and also exhibitssignificantly lower flushing requirements, resulting in lower levels oforganic extractables released from the depth filter media afterflushing.

BACKGROUND OF THE INVENTION

Depth filtration is commonly used in the clarification of cell cultures.As its name implies a depth filter utilizes its depth, or thickness, tocarry out filtration. The filter is typically a material structured witha gradient density, generally having more large pores near the top andsmaller pores at the bottom. Depth filters, unlike absolute filters,retain particles throughout the porous media, allowing for retention ofparticles both larger and smaller than the pore size. Particle retentionis thought to involve both size exclusion and adsorption throughhydrophobic, ionic and other interactions. Fouling mechanisms of a depthfilter may include pore blockage, cake formation and/or poreconstriction.

In many cases, depth filters can be run in series such that most of thecoarser particles are removed during the first filtration stage andfiner particles are filtered out in a second stage. Thus, in a cellculture where there is a broad distribution of particle sizes such asfrom cells and cell debris, depth filters are intended to retain themajority of suspended particulates.

Traditional depth media is composed of (1) cellulose, (2) diatomaceousearth (DE) or other filter aids, and (3) a wet-strength resin. However,these materials can contain trace amounts of beta glucans, metals andbioburden which can be extracted into the aqueous process stream.

In the biotech industry, these contaminants are undesirable and couldpose potential interference with purification schemes, as well as havenegative interactions with the product molecule or exceed typicalacceptance criteria established by the industry. For example, it hasbeen shown that material extracted from cellulose fiber filters, whichwas later identified as beta glucan, resulted in false positives forendotoxins in Limulus Amebocyte Lysate (LAL) testing (Pearson, F. C., etal 1984 Applied and Environmental Biology 48:1189-1196). In some cases,high levels of aluminum ions in the final product can have a neurotoxiceffect on the human nervous system.

Prior to use, depth filters require extensive preflushing, usually withan aqueous solution such as water, to reduce the levels of organic andinorganic contaminants to an acceptable value.

To reduce bioburden, depth filters can be pretreated with a causticsanitant such as 0.5 N NaOH for 30 min.

Another method for reducing depth filter bioburden is to subject thedepth filter to radiation treatment such as gamma irradiation.

Still another method for reducing depth filter bioburden is byautoclaving or steam-in-place, in which the entire filter devicecontaining the depth filter component is subjected to steam under highpressure. While these methods may reduce bioburden, they often have anegative impact on extractables.

Furthermore, diatomaceous earth (DE), a naturally-occurring material, isthe primary source of metal extractables, and because DE is anaturally-occurring material its composition is subject to largevariability depending on where the DE is mined. While pretreatment of DEwith acid can reduce the metal extractables, it also adds an extraprocessing step. Other silica-based filter aids such as perlite or sandare also limited in this respect.

Activated carbon filter aids are usually sourced from natural materialssuch as wood or coconut shell and again, and its composition is alsosubject to considerable variation.

Extractables from cellulose/DE depth filters can also be derived fromthe materials of construction themselves, such as from shedding offibers or particles during filtration. While the wet strength resingenerally helps to “glue” or adhere together the fibers and DE, there isinevitably some portion of particulates that are easily released fromthe depth filter. Indeed, sheets of cellulose/DE media can produce acloud of particulates from the simple action of fanning the filter sheetback and forth.

Filtration is limited by the available volume for particulates toaccumulate, i.e., the dirt holding capacity. Traditional depth filterstend to have low dirt holding capacity as much of the volume of thefilter is occupied by the fibers and filter aid. The depth filter mediacan also become rapidly plugged and lead to a buildup of a cake layer.

In addition, currently available depth filter media is not particularlywell-suited for the removal of soluble impurities, such as DNA and hostcell proteins, in processing cell culture/biological feedstreams. Suchcontaminants may interfere with the subsequent downstream purificationsteps including protein A affinity capture and bind/elute ion exchangechromatography steps. These impurities may significantly reduce productbinding capacity and limit the operational lifetime of thechromatography media. Higher impurity loads may also require theintroduction of additional flow-through polishing steps, expensivemembrane adsorbers or columns packed with anion exchange resins tofurther reduce the impurity load to within acceptable levels.

SUMMARY OF THE INVENTION

In response to the above needs and problems associated with depth filtermedia, the present invention avoids extensive preflushing needs andrelease of organic, inorganic and bioburden extractables by providing adepth filter media having a reduced amount of extractables in thefilters, thereby reducing the amount of water required for pre-useflushing, and exhibits an increased binding capacity for host cellproteins and other soluble impurities within a cell culture/biologicalfeedstream during a flow through adsorption process for harvested cellculture fluids.

Another object of this invention is to provide a composite depth filtermedia comprising a nonwoven first layer integral with a second layercomprising (1) fibers, (2) a filter aid, (3) and a wet strength resinhaving a reduced amount of organic, inorganic and bioburdenextractables, thereby reducing the amount of water required for pre-useflushing.

A further object of this invention is to provide a depth filter mediacomprising (1) nonwovens including polypropylene, polyesters,polyethylene, nylon, polyacrylonitrile, carbon and glass, (2)fibrillated fibers including polyacrylonitrile or polyacrylonitrilecopolymer fibers having a Canadian Standard Freeness from about 10 mL to800 mL, (3) filter aids including silica, alumina, glass, metal oxidesor mixed-metal oxides, ion-exchange resins and carbon, and (4) wetstrength resins including water-soluble synthetic polymers comprise ureaor melamine-formaldehyde based polymers,polyaminopolyamide-epichlorohydrin (PAE) polymers and glyoxalatedpolyacrylamide (GPAM) resins.

Another object of this invention is to provide depth filter media havingreduced shedding of particulates.

A further object of this invention is to provide a depth filter mediawith improved chemical or radiation resistance with lower flushingvolumes than conventional depth filter media.

A further object of this invention is to provide entirely syntheticfilter media with high dirt holding capacity and excellent retention ofcoarse\medium and fine particulates.

Another object of this invention is to provide a depth filter mediahaving an increased binding capacity for soluble process impuritieswithin a biological product feedstream. These soluble process impuritiesmay include host cell proteins (HCP) and DNA. Such depth filter mediapermits a low level of host cell protein and DNA impurity clearance fromharvested cell culture fluid (HCCF) feedstreams.

Another object of this invention is to provide a depth filter media thataccomplishes this low level of impurity clearance by using a flowthrough adsorption process for soluble impurities which occurs alongsidethe secondary clarification of insoluble impurities, cellular debris,and colloidal matter.

Another object of this invention is to provide a depth filter media thatincorporates inorganic filter aids of sufficient surface area andsurface charge characteristics to bind a defined population of solubleprocess impurities, such as HCP and DNA, within the feedstream by acombination of ionic and hydrophobic adsorption mechanisms.

Additional features and advantages of the invention will be set forth inthe detailed description and claims, which follows. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art. It isto be understood that the foregoing general description and thefollowing detailed description, the claims, as well as the appendeddrawings are exemplary and explanatory only, and are intended to providean explanation of various embodiments of the present teachings. Thespecific embodiments described herein are offered by way of example onlyand are not meant to be limiting in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic embodiment of one example of the depth filtermedia according to the invention;

FIG. 2 depicts total organic carbon (TOC) flush out curves for filtersflushed with 100 L/m² water at 600 LMH; fractions collected atdesignated intervals for TOC analysis;

FIG. 3 depicts pressure profiles for filters loaded with clarifiednon-expressing CHOs cell culture at 100 LMH to 100 L/m²;

FIG. 4 depicts turbidity breakthrough curves for filters loaded withclarified non-expressing CHOs cell culture (173 NTU) at 100 LMH to 100L/m²; fractions collected at designated intervals;

FIG. 5 depicts DNA breakthrough curves for filters loaded with clarifiednon-expressing CHOs cell culture (91 μg/mL) at 100 LMH to 100 L/m²;fractions collected at designated intervals;

FIG. 6A are pressure profiles for coupled primary and secondaryclarification devices described in example 25 (2:1 area ratio forprimary:secondary clarification depth filters), in accordance withcertain embodiments of the invention;

FIG. 6B is a plot of HCP impurity breakthrough for coupled Millistak+primary and secondary depth filter benchmarks (D0HC/X0HC, black line)and coupled, prototype primary and secondary depth filters (device ID7-1/device ID 7-2, grey line), in accordance with certain embodiments ofthe invention;

FIG. 7A are pressure profiles for uncoupled secondary clarificationdevices described in example 25, in accordance with certain embodimentsof the invention; and

FIG. 7B is a plot of HCP and DNA impurity breakthrough for an uncoupledsecondary depth filter (ID 7-3, HCP: black line, DNA: black circles) andan uncoupled secondary depth filter (ID 7-2, HCP: grey line, DNA: greycircles) in accordance with certain embodiments of the invention.

DESCRIPTION OF THE EMBODIMENTS

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”whether or not explicitly indicated. The term “about” generally refersto a range of numbers that one would consider equivalent to the recitedvalue (i.e., having the same function or result). In many instances, theterm “about” may include numbers that are rounded to the nearestsignificant figure.

Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Moreover, allranges disclosed herein are to be understood to encompass all sub rangessubsumed therein.

Before describing the present invention in further detail, a number ofterms will be defined. Use of these terms does not limit the scope ofthe invention but only serve to facilitate the description of theinvention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyto the same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated by reference.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “bubble point pore size” or “BP” is the pore size of thelargest pore in the filter medium.

As used herein the phrase “cell culture” includes cells, cell debris andcolloidal particles, biomolecule of interest, HCP, and DNA.

The term “capture step”, as used herein, generally refers to a methodused for binding a target molecule with a chromatography resin, whichresults in a solid phase containing a precipitate of the target moleculeand the resin. Typically, the target molecule is subsequently recoveredusing an elution step, which removes the target molecule from the solidphase, thereby resulting in the separation of the target molecule fromone or more impurities. In various embodiments, the capture step can beconducted using a chromatography media, such as a resin, membrane ormonolith.

The terms “Chinese hamster ovary cell protein” and “CHOP” as usedinterchangeably herein, refer to a mixture of host cell proteins (“HCP”)derived from a Chinese hamster ovary (“CHO”) cell culture. The HCP orCHOP is generally present as an impurity in a cell culture medium orlysate (e.g., a harvested cell culture fluid containing a protein orpolypeptide of interest (e.g., an antibody or immunoadhesion expressedin a CHO cell). Generally, the amount of CHOP present in a mixturecomprising a protein of interest provides a measure of the degree ofpurity for the protein of interest. Typically, the amount of CHOP in aprotein mixture is expressed in parts per million relative to the amountof the protein of interest in the mixture.

The term “clarification step” or simply “clarification”, as used herein,generally refers to one or more steps used initially in the purificationof biomolecules. The clarification step generally comprises removal ofcells and/or cellular debris using one or more steps including any ofthe following alone or various combinations thereof, e.g.,centrifugation and depth filtration, tangential flow filtration,microfiltration, precipitation, flocculation and settling. In someembodiments, the present invention provides an improvement over theconventional clarification step commonly used in various purificationschemes. The clarification step generally involves the removal of one ormore undesirable entities and is typically performed prior to a stepinvolving capture of the desired target molecule. Another aspect ofclarification is the removal of soluble and insoluble components in asample which may later on result in the fouling of a sterile filter in apurification process, thereby making the overall purification processmore economical. The clarification step often includes a primaryclarification step(s) upstream from a secondary clarificationdownstream. The clarification of cell culture harvests and high-solidsfeedstocks from large harvest volumes from modern production batchbioreactors (<25,000 L) and high cell densities often require primary,as well as secondary clarification steps prior to any subsequentchromatography operations and the like.

The terms “coarse filtration” or “coarse/medium filtration”, as usedherein, generally refer to the removal of mostly whole cells and somecellular debris in the purification of biomolecules.

The term “fine filtration”, as used herein, generally refers to theremoval of mostly cellular debris, colloidal particles and solubleimpurities such as HCP, DNA, endotoxins, viruses and lipids in thepurification of biomolecules.

The term “column volume” or “CV” as used herein refers to the volume ofliquid equivalent to the volume of filter media. The volume of filtermedia may be calculated by the product of the surface area and thethickness of the filter.

Filter throughput values are generally expressed in terms of“liters/square meter” or “L/m²” though for equivalent comparisons,“column volume” or “CV” is used to account for large differences ofthickness between samples.

The terms “contaminant”, “impurity”, and “debris”, are usedinterchangeably herein, refer to any foreign or objectionable material,including a biological macromolecule such as a DNA, an RNA, one or morehost cell proteins (HCPs or CHOPs), endotoxins, viruses, lipids and oneor more additives which may be present in a sample containing a proteinor polypeptide of interest (e.g., an antibody) being separated from oneor more of the foreign or objectionable molecules using a depth filteraccording to the present invention.

It is understood that where the host cell is another mammalian celltype, E. coli, yeast cell, insect, or plant, HCP refers to the proteins,other than target proteins, found in a lysate of the host cell.

The term “mean flow pore size” or “MFP” as used herein is the porediameter at a pressure drop at which the flow through a wetted filtermedium is 50% of the flow through the dry filter medium.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

As used herein the term “organic extractable(s)” refers to contaminantsthat in the presence of water or other aqueous solutions used duringflushing, can potentially migrate or be extracted from materials used tomake filter media or membranes, such as porous depth filter media. Thesecontaminants may also include the materials of construction themselveswhich could potentially shed from the filter during flushing.

As used herein the phrase “low or lower organic extractable media”refers to a media that when extracted or flushed with water results inthe removal of extractables that can migrate from a material into asolvent including water under exaggerated conditions of time andtemperature.

The term “total organic extractable(s)” and “TOC” refers to themeasurement of organic molecules present in an aqueous solution such aswater and measured as carbon content. Analytical techniques used tomeasure TOC typically involve oxidation of all organic molecules insolution to carbon dioxide, measuring the resultant CO₂ concentration,and correlating this response to a known carbon concentration.

The term “parts per million” or “ppm” are used interchangeably herein.

Pore size ratings are usually given as a nominal value. In some cases,manufacturers provide a mean flow pore (MFP) size or a bubble point (BP)pore size. Both the MFP and BP can be measured using a capillary flowporometer.

The terms “target molecule”, “target biomolecule”, “desired targetmolecule” and “desired target biomolecule,” are used interchangeablyherein, and generally refer to a polypeptide or product of interest,which is desired to be purified or separated from one or moreundesirable entities, e.g., one or more impurities, which may be presentin a sample containing the polypeptide or product of interest.

As used herein the term “throughput” means the volume filtered through afilter.

As used herein the term “dirt holding capacity” is equivalent to filterthroughput of a given cell culture fluid, either from direct harvest orpreviously clarified. Higher throughput represents higher dirt holdingcapacity.

The depth filter of the present invention comprises components (A)fibers, (B) filter aid, (C) wet strength resin and (D) a nonwoven. Thecombination of these components in various configurations yields depthfilters with low extractables, high dirt holding capacities, goodchemical and/or radiation resistance, and an increased binding capacityfor host cell proteins and other soluble impurities contained inbiological product-containing feedstreams

Filter Materials

Component A. Fiber materials for use in depth filters have been widelydisclosed. Non-cellulose based materials include microglass fibers and avariety of synthetic polymers such as polypropylene and polyesters.Especially useful are fibrillated fibers, fibers which have beenprocessed to produce more surface area and a branched structure.Suitable fibrillated fibers include polyacrylonitrile or copolymers withpolyacrylonitrile, polyethylene, polypropylene and Vectran, by KurarayCo., Ltd. an aromatic polyester based fiber, either singly or incombination.

In preferred embodiments, fibers made from polyacrylonitrile (PAN)copolymers (Sterling Fibers Inc., Pace, Fla., USA) are used.

The degree of fibrillation of the fiber effects the Canadian StandardFreeness (CSF) or the drainage rate for a dilute suspension of thefibers. For example, more highly fibrillated fibers tend to have a lowerCSF. The preferred CSF ranges from 10 mL to 800 mL; in some embodiments,a range of 600 mL to 750 mL is used. In other embodiments, a range of200 mL to 600 mL is preferred. In still other embodiments, a range of 50mL to 300 mL is preferred. In yet other embodiments, fibrillated fiberswith different CSF can be combined to produce an average CSF in therange of 10 mL to 800 mL.

Component B. Filter aids can be particles provided in a variety ofshapes, sizes, and materials. For example, filter aid particles can bespherical, fibrous, plate-like or irregular. Further, the particles maybe milled, ground, blended or processed in other ways known in the artto produce smaller particles of irregular shape. As with the shape ofthe particles, the size of the filter aid need not be a single value. Itis desirable to have a distribution of particle sizes in the filter.

Processing, such as sieving or classification, can be done to size theparticles into fractions of narrower particle size distributions.Generally, the size of the filter aid particles may range from about0.01 μm to about 5 mm, preferably from about 10 μm to about 500 in someembodiments, from about 40 μm to about 200 μm in other embodiments, fromabout 0.1 μm to about 50 μm in still other embodiments, and from about0.01 μm to about 50 μm in yet other embodiments.

The filter aid may be porous, having interconnected porosity orclosed-cell porosity, or nonporous. Especially in the case ofclosed-cell porosity materials, if the particles are processed bymilling, blending or the like to produce smaller particles, the closedpores could be opened to reveal the porosity and the particle wouldessentially become nonporous.

Examples, of synthetic filter aids which can be used include silica,alumina, glass, other metal oxides or mixed-metal oxides, ion-exchangeresins and carbon. These materials can also be surface-modified bymethods known to those skilled in the art to impart a charge,hydrophobic or other functionality.

Inorganic filter aids having a sufficient surface area and surfacecharge characteristics bind to a defined population of soluble processimpurities, such as HCP and DNA, within the feedstream by a combinationof ionic and hydrophobic adsorption mechanisms.

Examples of suitable silica filter aids include, but are not limited to,precipitated silicas, silica gel and fumed silicas. In certainembodiments, the preferred silica filter aids are preferably selectedfrom precipitated silicas such as Sipernat® (Evonik Industries AG,Hanau-Wolfgang, Germany) or silica gels such as Kieselgel 60 (MerckKGaA, Darmstadt, Germany).

Alumina comes in many forms: porous, nonporous, acidic pH, neutral pH,basic (alkaline) pH, etc. In certain embodiments, the preferred aluminafilter aid embodiment is porous and with a basic pH, such as Merck KGaA,Darmstadt Germany aluminum oxide 150 basic.

Examples of glass filter aids include controlled pore glass, e-glass andexpanded glass. The preferred glass filter aid embodiment is expandedglass, and more preferably, expanded glass made from recycled glass,such as Poraver®, (Poraver North America Inc., Ontario, Canada).

Suitable ion-exchange resins are porous and rigid and preferably do nonot swell or shrink significantly in the presence or absence of water.The preferred ion-exchange resin embodiment is preferably positivelycharged.

Examples of carbon include activated carbon spheres or fibers derivedfrom rayon or other synthetic source.

Filter aids can be used singly or in combination so long as they producethe particle size ranges described above. The content by weight relativeto the total weight of fiber and filter aid can range from 0% to about90%, in some embodiments, from about 40% to about 80%.

Component C. Wet strength resins are known in the art. They arewater-soluble synthetic polymers with anionic and/or cationic groupsused to impart strength to a material when wet. Suitable wet strengthresins are urea- or melamine-formaldehyde based polymers,polyaminopolyamide-epichlorohydrin (PAE) polymers and glyoxalatedpolyacrylamide (GPAM) resins. Commercial resins are readily availablefrom Ashland, Inc. (formerly Hercules Inc.), The Dow Chemical Company,BASF Corporation and Georgia-Pacific Chemicals LLC. The content byweight of the wet strength resin based on the total weight of the fiberand filter aid ranges between about 0.5% and 5%, preferably between 1%and 3%.

Component D. Nonwovens are widely available in different materials,fiber diameters, basis weights, thicknesses and pore size ratings. Theycan be produced by various technologies such as meltblown, airlaid,spunbond, spunlace, thermal bond, electrospinning and wetlaid. Nonwovenscan be made from polymers, inorganics, metallics or natural fibers.Suitable materials include polypropylene, polyesters, polyethylene,nylon, polyacrylonitrile, carbon and glass. Depending on the desiredproperties, fiber diameters can range from about 1 nm to about 1 mm. Ina preferred embodiment, fiber diameters range from about 10 nm to about30 μm. The basis weight is defined as the weight of a material per givenarea. Generally, basis weight ranges from 5 to 350 g/m².

In a preferred embodiment, the basis weight ranges from 20 to 300 g/m².The thickness of the nonwoven can vary from 50 μm to about 1 cm. In apreferred embodiment, the thickness of the nonwoven is about 0.1 to 0.3cm.

In another embodiment, the thickness of the nonwoven is about 100 μm toabout 500 μm.

In still other embodiments, several layers of a nonwoven may be stackedtogether to achieve a thickness in the range of 200 μm to 1,000 μm.

The filter Components (A) through (D) are combined in variousconfigurations to make a depth filter having a gradient density porestructure.

In a preferred embodiment, the filter media are arranged such that thepore size rating of each layer is gradually reduced (i.e., pores sizerating gets smaller from top (i.e., upstream side of media) and tobottom (i.e., downstream side of media) of the filter media), whereinthe feed flow direction is typically from top to bottom of the filtermedia as well.

The following examples are provided for the purpose of furtherillustrating the present invention but are in no way to be taken aslimiting. In addition, the following examples are provided so as toprovide those of ordinary skill in the art with a complete disclosureand description of how to make and how to practice the methods of theinvention, and are not intended to limit the scope of what the inventorregards as his invention. Efforts have been made to insure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.), but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, temperature is in degree Celsius (° C.), chemicalreactions were performed at atmospheric pressure or transmembranepressure, as indicated, the term “ambient temperature” refers toapproximately 25° C. and “ambient pressure” refers to atmosphericpressure.

Unless otherwise specifically provided herein, the following methods,materials, processes, and conditions provided in sections (I) through(IV) below, were used in the practice of various embodiments of theinvention, and are intended to be exemplary of the invention:

I. Layer Configuration

In the following examples, and as schematically depicted in FIG. 1 ,each filter in these embodiments contains up to three (3) componentlayers: where Layer zero (0) is a nonwoven or stacked multiple sheets ofa nonwoven to give the desired thickness, and Layers one (1) and two (2)may be the same, but can optionally be made of similar or differentmaterials of similar or different composition.

II. Handsheet Formation

Generally, fiber, water, and wet strength resin, if used, are processedin a readily available blender (Blendtec Corporation, Orem, Utah, USA).Filter aid is then blended in. The slurry is filtered onto a meshsupport by gravity draining. Residual water is removed by vacuumfiltration and drying at 105° C. for 1 to 2 h.

III. Process Scale Filter Media Formation

Numerous methods of forming fiber and filter aid into depth filter mediaon the processing scale are known in the art: air-laying, melt-pressing,mechanical compression and wet-laying. The preferred process for makingdepth filter media for Layers 1 and 2 is the wet-laid process: allcomponents are dispersed in water to form a well-mixed slurry. Theslurry is applied onto a moving belt where water is allowed to drain ora vacuum is applied to remove excess water. The subsequent pad formedtravels along the belt through a series of ovens with adjustabletemperature zones for drying. Preferably, the temperature zones rangefrom 80° C. to about 250° C. Optionally, the media may also undergocompression through a series of rollers during heating to adjust forthickness. Preferably, the thickness of the media lies between 0.1 cmand 0.5 cm.

IV. Filter Assembly

The filter is assembled according to step A, stacked together so thatLayer 0 precedes Layer 1 and Layer 1 precedes Layer 2. In the caseswhere Layer 0 is not used, Layer 1 precedes Layer 2. Layer 1 and Layer 2may also be used individually.

The layer(s) are preferably housed inside a filter cell, reusable ordisposable, such that each layer is in contact with the preceding layerand there is sufficient and minimal headspace for the challenge fluid touniformly pass through the filter.

The following examples are provided so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the compositions of the invention and how to practice the methodsof the invention and are not intended to limit the scope of what theinventor regards as his invention.

Efforts have been made to insure accuracy with respect to numbers used(e.g. amounts, temperature, etc.), but some experimental errors anddeviation should be accounted for. Unless indicated otherwise,temperature is in degrees C., chemical reactions were performed atatmospheric pressure or transmembrane pressure, as indicated, the term“ambient temperature” refers to approximately 25° C. and “ambientpressure” refers to atmospheric pressure. The invention will be furtherclarified by the following examples which are intended to be exemplaryof the invention.

Description of the Analytical Methods Used in the Examples.

(i) Water Flow Rate Test

The water flow rate of samples (23 cm²) is measured at 10 psi.

(ii) Extractables Flush Test

Samples (23 cm²) are flushed with water at 600 LMH to 100 L/m².Fractions are collected at predetermined intervals for TOC analysis.

(iii) Caustic Sanitization

Samples (23 cm²) are flushed with 0.5 N NaOH at 100 to 300 LMH for 30min. Samples are optionally subsequently flushed with water withfractions collected at predetermined intervals for TOC analysis. Filtersare then equilibrated with 100 mM phosphate buffer pH 7.

(iv) Throughput and Retention

Samples (23 cm²) are loaded with a cell culture feed stream or affinitycapture pool at 100 LMH until the pressure drop across the filterreaches 20 psid. Filtrate fractions are collected at designatedintervals, typically 5 minutes, and measured for turbidity; in somecases, fractions are also assayed for HCP, DNA, and/or mAbconcentration.

EXAMPLES Example 1

Depth filter compositions, for fine filtration, each having a two (2)layer configuration according to an embodiment of the invention include:

Filter 1A. polyacrylonitrile (PAN)/silica

Layer 1: 5.06 g PAN (Sterling Fibers CFF® 111-3 fibrillated pulp,CSF=250 mL), 0.38 g polyaminopolyamide-epichlorohydrin resin (Wetstrength resin C®), 304 mL water, and 3.22 g silica (Sipernat® 120Evonik Corporation, Parsippany, N.J., USA)

Layer 2: same as Layer 1

Blending cycle: 30 s on Soups preset, then 10 Pulses

Filter 1B. polyacrylonitrile (PAN)/glass

Layer 1: 8.16 g PAN fibers (Sterling Fibers CFF 114-3, CSF=60 mL), 2.72g PAN fibers (EFTec™ nanofibrillated fibers A-010-4, CSF=10 mLEngineered Fibers Technology, Shelton, Conn.), 0.48 gpolyaminopolyamide-epichlorohydrin resin (Wet strength resin C®), 330 mLwater, 3.03 g glass (Poraver® 0.040-0.125 mm, milled to average particlesize 12 μm)

Layer 2: 2.72 g PAN fibers (Sterling Fibers CFF 114-3), 8.16 g PANfibers (EFTec A-010-4), 0.48 g polyaminopolyamide-epichlorohydrin resin(Wet strength resin C®), 330 mL water, 3.03 g glass (Poraver 0.040-0.125mm, milled to average particle size 12 μm)

Blending cycle: 25 s on Soups preset, then 25 Pulses

Filter 1C. polyacrylonitrile (PAN)/ion exchange (IEX) bead

Layer 1: 6.33 g PAN fibers (Sterling Fibers CFF 114-3), 330 mL water,4.03 g IEX beads (Reillex HPQ™ Polymer, milled to average particle sizeof 6.5 μm, Vertellus Specialties, Inc., Indianapolis, Ind., USA)

Layer 2: 3.16 g PAN fibers (Sterling Fibers CFF 114-3), 3.16 g PANfibers (EFTec A-010-4), 330 mL water, 4.03 g IEX beads (Reillex HPQ,milled to average particle size of 6.5 μm)

Blending cycle: 25 s on Soups preset, then 25 Pulses

Filter 1D. polyacrylonitrile (PAN)/diatomaceous earth (DE)

Layer 1: 4.21 g PAN (Sterling Fibers CFF 111-3), 1.40 g PAN fibers(Sterling Fibers CFF 114-3), 0.27 g polyaminopolyamide-epichlorohydrinresin (Wet strength resin C®), 330 mL water, and 3.58 g diatomaceousearth (MN-4/Celite® 507 in a 1:1 ratio Imerys Filtration Minerals Inc.,San Jose, Calif., USA)

Layer 2: 1.40 g PAN (Sterling Fibers CFF 111-3), 4.21 g PAN fibers(Sterling Fibers CFF 114-3), 0.30 g polyaminopolyamide-epichlorohydrinresin (Wet strength resin C®), 330 mL water, and 3.22 g diatomaceousearth (MN-4/Celite® 507 in a 1:3 ratio)

Blending cycle: 15 s on Speed 3 preset, then 10 s on Speed 1 preset

Filter 1E. polyacrylonitrile (PAN)/alumina

Layer 1: 6.53 g PAN (Sterling Fibers CFF 114-3), 2.18 g PAN fibers(EFTec A-010-4), 0.38 g polyaminopolyamide-epichlorohydrin resin (Wetstrength resin C®), 330 mL water, and 4.83 g alumina (Merck KGaA, milledto an average particle size of 12 μm)

Layer 2: 2.18 g PAN (Sterling Fibers CFF 114-3), 6.53 g PAN fibers(EFTec A-010-4), 0.38 g polyaminopolyamide-epichlorohydrin resin (Wetstrength resin C®), 330 mL water, and 4.83 g alumina (Merck KGaA, milledto an average particle size of 12 μm)

Blending cycle: 15 s on Speed 3 preset, then 10 s on Speed 1 preset

For comparison purposes, a conventional cellulose/diatomaceous depthfilter, Millistak X0HC, is also presented.

Characterization of the Depth Filters

Water flow rate (WFR) @10 psi Thickness Filter (L/min/m²) (cm) 1A 100.75 1B 9 0.79 1C 20 0.76 1D 21 0.76 1E 13 0.79 X0HC control 8 0.75

Example 2

Depth filter media for coarse filtration according to an embodiment ofthe invention was prepared on a conventional wet laid media productionline using PAN (Sterling Fibers CFF 106-3, CSF=600 mL) and 2.5%polyaminopolyamide-epichlorohydrin resin (Wet strength resin C®).Samples denoted PAN25 (23 cm² cutouts) had a basis weight of 711 g/m², athickness of 0.40 cm and a water flow rate of 2038 L/min/m². DSF

Example 3

Depth filter composition, for coarse/medium filtration, having a two (2)layer configuration according to an embodiment of the invention include:

Filter 3A. polyacrylonitrile (PAN)/glass

Layer 1: PAN25, as prepared in Example 2

Layer 2: 5.06 g PAN fibers (Sterling Fibers CFF 106-3), 0.75 gpolyaminopolyamide-epichlorohydrin resin (Wet strength resin C®), 300 mLwater, 3.22 g glass (Poraver 1-2 mm, milled to average particle size 26μm)

Blending cycle: 30 s on Soups preset, then 10 Pulses

For comparison purposes, a conventional cellulose/diatomaceous earthdepth filter, Millistak+® D0HC, is also presented.

Characterization of the Depth Filter

Thickness Filter (cm) 3A 0.74 D0HC control 0.74

Example 4

Depth filter composition, for coarse/medium filtration, having a three(3) layer configuration, according to an embodiment of the inventionincludes:

Filter 4A. Nonwoven/PAN/glass

Layer 0: mixed synthetic fiber nonwoven (Hollingsworth & Vose, EastWalpole, Mass., USA) having 215 g/m² basis weight, 0.20 cm thickness

Layer 1: PAN 25, as prepared in Example 2.

Layer 2: same composition as in Example 3A

Characterization of the Depth Filters

Thickness Filter (cm) 4A 0.78

Example 5

Depth filter compositions, for fine filtration, each having a three (3)layer configuration, according to an embodiment of the inventioninclude:

Filter 5A. Nonwoven/PAN/IEX beads

Layer 0: polypropylene microfiber sheet (Hollingsworth & Vose EastWalpole, Mass., USA) having 20 g/m² basis weight, 0.1 mm thickness, 6.5mm mean flow pore diameter—two (2) sheets stacked together to make atotal thickness of 0.2 mm

Layer 1: same composition as in Example 1C

Layer 2: same composition as in Example 1C

Filter 5B. Nonwoven/PAN/glass

Layer 0: polypropylene microfiber sheet (Hollingsworth & Vose) having 20g/m² basis weight, 0.1 mm thickness, 6.5 mm mean flow pore diameter—two(2) sheets stacked together to make a total thickness of 0.2 mm

Layer 1: same composition as in Example 1B

Layer 2: same composition as in Example 1B

Characterization of the Depth Filters

Water flow rate (WFR) @10 psi Thickness Filter (L/min/m²) (cm) 5A 150.85 5B 9 0.79

Example 6

Filter 1A in Example 1 was subjected to an extractables flush andthroughput and retention testing with a non-expressing CHOs feed stock.Conventional (i.e., comparative) depth filter media Millistak+® X0HC wasalso tested for comparison.

TOC after 50 L/m² Throughput Turbidity Filter water flush (ppm) (L/m²)retention (%) 6A^(a) 0.49 82 99.7 X0HC^(a) 2.46 46 99.8 ^(a)filters wereonly loaded to 10 psid

Filter 6A demonstrates lower TOC extractables and higher throughput thanthe conventional X0HC, while retaining similar turbidity retentionvalues.

Example 7

To demonstrate feasibility on a manufacturing line, the composition fromfilter 1A in Example 1 was processed on conventional wet laid mediaproduction equipment. Sheets produced had a basis weight of 1130 g/m², athickness of 0.43 cm and a water flow rate of 53 L/min/m². Anextractable flush was done as provided herein.

The samples were additionally subjected to throughput and retentiontesting as provided herein with a primary-clarified non-expressing CHOsfeedstock.

TOC after 50 L/m² Throughput Turbidity Filter water flush (ppm) (L/m²)retention (%) 7A^(a) 1.19 80 99.8 X0HC^(b) 2.98 55 99.9 ^(a)filter wasonly loaded to 8.0 psid ^(b)filter was only loaded to 10 psid

Filter 7A demonstrates lower TOC extractables and higher throughput thanthe conventional X0HC, while retaining similar Turbidity retentionvalues.

Example 8

Filter 7A in Example 7 was subjected to gamma irradiation (25-40 kGy).An extractables flush was done for both irradiated and nonirradiatedsamples. The extractables increased after exposure to gamma butrelatively less so when compared to the conventional (i.e., comparative)depth media Millistak+® X0HC.

TOC after 50 L/m² Post-gamma, TOC after 50 L/m² Filter water flush (ppm)water flush (ppm) 7 1.31 4.28 X0HC 3.45 12.22

Example 9

Filter 1B in Example 1 was subjected to an extractables flush.Conventional depth filter, Millistak+® X0HC, was also tested forcomparison.

TOC after 50 L/m² TOC after 100 L/m² Filter water flush (ppm) waterflush (ppm) 1B 1.11 0.53 X0HC 4.42 1.16

Filter 1B demonstrates lower TOC extractables than the conventionalX0HC: only 50 L/m² water flush was needed for Filter 1B as compared to100 L/m² for X0HC to achieve a similar TOC value of ˜1.1 ppm.

Example 10

Filter 1B in Example 1 was subjected to caustic sanitization and testedfor throughput and retention with a non-expressing CHOs feed stockcentrate.

Flushed with water Sanitized with 0.5N NaOH prior to load prior to loadThroughput Turbidity Throughput Turbidity Filter (L/m²) retention (%)(L/m²) retention (%) 1B 198 97.7 222 97.0

Filter 1B demonstrates similar Throughput and Retention values with andwithout a pre-use caustic sanitization treatment step.

Example 11

Layer 1, in filter 1C in Example 1, was subjected to gamma irradiation(25-40 kGy). An extractables flush was done for both irradidated andnonirradiated samples. The extractables increased slightly afterexposure to gamma.

TOC after 50 L/m² Post-gamma, TOC after 50 L/m² Filter water flush (ppm)water flush (ppm) 1C Layer 1 0.89 2.53

Example 12

Filter 1C in Example 1 was also tested with a monoclonal antibodyfeedstock purified with a Protein A capture step for throughput andretention. The loading was 100 L/m². Host cell protein and DNA removalas well as product recovery in the pooled filtrate was also determined.Conventional depth filter, Millistak+® X0HC, was also tested forcomparison.

Turbidity HCP DNA mAb recovery Filter retention (%) (LRV) retention (%)(%) 1C 91.6 2.0 >97 97 X0HC 91.2 1.5 >97 91

Filter 1C demonstrates better HCP removal and higher product recoverythan the conventional X0HC, while retaining similar Turbidity retentionand DNA retention values.

Example 13

Filters PAN/DE and PAN/Alumina

Filters 1D and 1E in Example 1 were both subjected to causticsanitization followed by throughput and retention testing. Conventionaldepth filter, Millistak+® X0HC, was also tested for comparison.

Flushed with water Sanitized with 0.5N NaOH prior to load prior to loadThroughput Turbidity Throughput Turbidity Filter (L/m²) retention (%)(L/m²) retention (%) 1D 123 99.0 155 98.9 1E  155^(a) 98.8^(a)  156^(b)99.0^(b) X0HC 102 99.5  88 99.2 ^(a)filter was only loaded to 17 psid^(b)filter was only loaded to 11 psid

Example 14

Filter 3A in Example 3 was subjected to an extractables flush.Conventional depth filter, Millistak+D0HC, was also tested forcomparison.

TOC after 50 L/m² Filter water flush (ppm) 3A 0.46 D0HC 3.05

Filter 3A demonstrates lower TOC extractables than the conventionalD0HC.

Example 15

Layer 0 in filter 4A in Example 4 was subjected to gamma irradiation(25-40 kGy). An extractables flush was done for both irradidated andnonirradiated samples. There was no apparent change in the extractablesafter exposure to gamma.

TOC after 50 L/m² Post-gamma, TOC after 50 L/m² Filter flush (ppm) waterflush (ppm) 4A, Layer 0 0.02 0.02

Example 16

Filter 4A in Example 4 was subjected to throughput and retention testingwith a mAb feed stock.

Throughput Turbidity retention Filter (CV) (%) 4A 4.7 96.9 D0HC 3.5 90.1

Filter 4A demonstrates higher throughput and turbidity retention ascompared to the conventional D0HC.

Example 17

Filter 1C in Example 1 was subjected to an extractables flush.

Filter 5A in Example 5, and filter 1C in Example 1, were each subjectedto throughput and retention testing with a mAb feed stock previouslyprimary-clarified with Millistak+® D0HC. Filtrates were alsocharacterized for DNA retention. For comparative purposes, conventionalcellulose/diatomaceous earth media, Millistak+® X0HC, was also tested.The results are summarized and displayed in FIGS. 2 to 5 .

Throughput Turbidity retention DNA retention Filter (CV) (%) (%) 1C^(a)11.7 98.3 99.9 5A^(b) 10.6 97.4 99.9 X0HC 8.8 95.2 95.3 ^(a)filter wasonly loaded to 5.3 psid ^(b)filter was only loaded to 7.6 psid

The test results depicted in FIGS. 2 to 5 demonstrate:

1. Filter 1C (PAN/IEX beads) not only has a total overall lower TOC thanthe conventional filter X0HC, but the extractables profile starts offlower and ends lower.

2. Filters 1C (PAN/IEX beads) and 5A (nonwoven/PAN/IEX beads) have lowerpressure profiles than X0HC. Indeed, the X0HC filter reaches 20 psiwhile the filters of the current invention stay well below 10 psi. Thisresults in a significantly higher throughput of feed stock that can beprocessed through the filters of the current invention.

3. Filters 1C (PAN/IEX beads) and 5A (nonwoven/PAN/IEX beads) have lowerturbidity profiles than X0HC. The filters of the current invention canbe said to have higher turbidity retention. Significant turbiditybreakthrough did not occur up to 100 L/m² in Filters 1C and 5A, ascompared to some small breakthrough in X0HC at ˜40 L/m². Continuedloading of the filters may still provide good retention.

4. Filters 1C (PAN/IEX beads) and 5A (nonwoven/PAN/IEX beads) have lowerDNA profiles than X0HC. The filters of the current invention can be saidto have higher DNA retention. Significant DNA breakthrough did not occurup to 100 L/m² in Filters 1C and 5A, as compared to some smallbreakthrough in X0HC at ˜50 L/m². Continued loading of the filters maystill provide good retention.

Example 18

To further illustrate the advantages of PAN as compared to cellulose,all-fiber pads were formed using a 1% fiber in water slurry. The padswere dried at 105° C. for 2 h. Subsequently, each pad was immersed inwater for several hours under agitation. The cellulose redispersed intoloose fibers, while the PAN remained as a pad with no observable loosefibers.

In following Examples 19 to 27, under Component B—filter aid, the silicagel particle size ranges are fractions of commercially available SilicaGel 60, manufactured by Merck KGaA (Darmstadt, Germany), having a poresize of about 60 A (6 nm). The silica particles used in certainembodiments of the invention in these Examples were isolated by asieving operation, wherein the first sieving fraction, labeled “finesilica particles”, resulted in small/fine silica particles having aparticle size≤(less than or equal to) about 5 microns, and the secondsieving fraction, labeled “coarse silica particles”, resulted inlarge/coarse silica particles having a particle size≤(less than or equalto) about 40 μm.

TABLE 1 Table of depth filter media formulations used in the followingexamples. Depth Component A - Component B - Component C - filter fibermaterial filter aid wet strength resin media ID (% loading) (% loading)(% loading) 1-1 PAN 106 (42%) coarse silica WET STRENGTH particles (58%)RESIN X (2%) 1-2 PAN 111 (42%) coarse silica WET STRENGTH particles(58%) RESIN X (2%) 1-3 PAN 106 (21%) coarse silica WET STRENGTH PAN 111(21%) particles (58%) RESIN X (2%) 1-4 PAN 106 (50%) — wet strength PAN111 (50%) resin Z (3%) 1-5 PAN 106-2 (42%) coarse silica wet strengthparticles (58%) resin Z (3%) 1-6 PAN 106 (50%) — wet strength PAN 111(50%) resin Y (1%) 1-7 PAN 106-2 (42%) coarse silica wet strengthparticles (58%) resin Y (1%) 1-8 PAN 106-2 — WET STRENGTH (100%) RESIN X(2%) 1-9 PAN 106 (21%) coarse silica wet strength PAN 111 (21%)particles (58%) resin Z (3%) 1-10 PAN 106 (100%) — wet strength resin Y(1%) 2-1 PAN 111 (32%) Sipernat 120 WET STRENGTH (68%) RESIN X (2%) 2-2PAN 111 (32%) Sipernat 120 — (68%) 2-3 PAN 114 (32%) fine silica WETSTRENGTH particles (34%) RESIN X (2%) coarse silica particles (34%) 2-4PAN 114 (32%) fine silica WET STRENGTH particles (51%) RESIN X (2%)coarse silica particles (17%) 3-1 PAN 114 (46%) fine silica — particles(27%) coarse silica particles (27%) 3-2 PAN 114 (26%) fine silica —particles (37%) coarse silica particles (37%) 3-3 PAN 114 (46%) finesilica — particles (54%) coarse silica particles (0%) 3-4 PAN 114 (26%)fine silica — particles (74%) coarse silica particles (0%) 3-5 PAN 114(46%) fine silica WET STRENGTH particles (27%) RESIN X (4%) coarsesilica particles (27%) 3-6 PAN 114 (26%) fine silica WET STRENGTHparticles (37%) RESIN X (4%) coarse silica particles (37%) 3-7 PAN 114(46%) fine silica WET STRENGTH particles (54%) RESIN X (4%) coarsesilica particles (0%) 3-8 PAN 114 (26%) fine silica WET STRENGTHparticles (74%) RESIN X (4%) coarse silica particles (0%) 3-9 PAN 114(37%) fine silica WET STRENGTH particles (47%) RESIN X (2%) coarsesilica particles (16%) comparative cellulose (42%) diatomaceous —example 4-1 earth (DE1) tight (29%) diatomaceous earth (DE2) open (29%)comparative cellulose (22%) DE1 (39%) — example 4-2 DE2 (39%)comparative cellulose (42%) DE1 (58%) — example 4-3 DE2 (0%) comparativecellulose (22%) DE1 (78%) — example 4-4 DE2 (0%) comparative cellulose(42%) DE1 (29%) WET STRENGTH example 4-5 DE2 (29%) RESIN X (4%)comparative cellulose (22%) DE1 (39%) WET STRENGTH example 4-6 DE2 (39%)RESIN X (4%) comparative cellulose (42%) DE1 (58%) WET STRENGTH example4-7 DE2 (0%) RESIN X (4%) comparative cellulose (22%) DE1 (78%) WETSTRENGTH example 4-8 DE2 (0%) RESIN X (4%) comparative cellulose (32%)DE1 (51%) WET STRENGTH example 4-9 DE2 (17%) RESIN X (2%) 5-1 PAN 114(10%) fine silica WET STRENGTH PAN 111 (32%) particles (29%) RESIN X(2%) coarse silica particles (29%) 5-2 PAN 106 (21%) coarse silica WETSTRENGTH PAN 111 (21%) particles (58%) RESIN X (2%) 5-3 PAN 106 (41%)coarse silica WET STRENGTH particles (59%) RESIN X (2%) 6-1 PAN 106(21%) coarse silica wet strength PAN 111 (21%) particles (58%) resin Z(3%) comparative PAN 106 (100%) — wet strength example 6-2 resin Y (1%)comparative cellulose (42%) diatomaceous wet strength example 6-3 earth(DE3) resin Z (3%) (53%) comparative cellulose (100%) — wet strengthexample 6-4 resin Y (1%)

Example 19

General Procedure Used for Static Binding Capacity Measurements.

Six grams of a depth filter media were suspended in 300 mL water andblended to form a dilute fiber slurry. The suspension was transferred toa 500 mL Nalgene® bottle using an additional 200 mL of water forrinsing. A 10 mL aliquot of the fiber suspension was transferred to apre-weighed 15 mL centrifuge tube. The centrifuge tube was spun for 5minutes in a bench-top centrifuge to pellet the fiber solids. Thesupernatant was removed by means of a pipette and 10 mL of either a 1g/L BSA or 1 g/L myloglobin solution in 25 mM Tris pH 7.3 were added.Alternatively, for host cell protein static binding capacitymeasurements, a 10 mL aliquot of harvested cell culture fluid that hadbeen centrifuged and sterile filtered through a 0.2 μm MilliporeExpress® membrane (EMD Millipore, Billerica, Mass.) was used. The fibersuspension was then agitated at room temperature for 18 hours. Thecentrifuge tube was then spun for 5 minutes in a bench-top centrifuge topellet the fiber solids. For the protein static binding capacitymeasurements, a sample of the supernatant solution was taken for UV-vismeasurement at 280 nm (for BSA) or 409 nm (for myoglobin) and the changein protein concentration from the feed sample was determined.

Alternatively, for host cell protein static binding capacitymeasurements, a 1 mL aliquot of the supernatant solution was taken forHCP ELISA assay. The remaining supernatant solution was then removedfrom the centrifuge tube and the damp material was dried in an oven at60° C. for 18 to 36 hours. The final weight of the dry depth filtermedia was determined and this value was used to calculate the staticbinding capacity of the depth filter media by dividing the amount ofadsorbed protein (or HCP) by the weight of the depth filter media. Theobtained value is the static binding capacity in terms of mg (protein)/g(depth filter media).

Example 20

Static binding capacity measurements of selected depth filter mediaformulations.

BSA and myoglobin static binding capacity measurements were performedfor various depth filter media formulations according to the methoddescribed in Example 19.

The static binding capacities for these samples are provided in Table 2.

The data in Table 2 shows that the BSA static binding capacities arecomparable for all depth filter media formulations, regardless of PANfiber type, silica loading, or resin type. In contrast, coarse silicaparticle (having a particle size less than or equal to about 40 μm)filter aides had an unexpectedly strong effect on the myoglobin staticbinding capacity. The four depth filter media formulations which lackthe coarse silica particle filter aid gave no myoglobin SBC(compositions 1-4, 1-6, 1-8, 1-10), while the other six formulationsdemonstrate a high myloglobin SBC of approximately 30 mg/g (compositions1-1 thru 1-3, 1-5, 1-7, and 1-9).

At the application pH of 7.3, myoglobin is largely uncharged(isoelectric point=6.8-7.2) and BSA is negatively charged (isoelectricpoint≈5). Under such conditions, modest BSA static binding capacitiesfor the depth filter media formulations may occur by way ofelectrostatic interactions between the positively charged binder resincomponent and the negatively charged BSA. Under these same conditions,strong hydrophobic interactions may occur between the coarse silicaparticles and the uncharged myoglobin protein. While not wishing to bebound to any theory, it is alleged that the increased myoglobin staticbinding capacity may be attributed to the relatively large surface areafor the coarse silica particle filter aid used in these depth filtermedia formulation embodiments.

TABLE 2 BSA and myoglobin SBC for selected depth filter mediaformulations. Depth Filter BSA SBC Myoglobin SBC Media ID (mg/g) (mg/g)1-1 8 33 1-2 8 31 1-3 6 32 1-4 5 0 1-5 6 27 1-6 7 1 1-7 8 28 1-8 7 1 1-99 29 1-10 5 1

Example 21

Static Binding Capacity Measurements of Selected Depth Filter MediaFormulations.

BSA, myoglobin, and HCP static binding capacity measurements wereperformed for various depth filter media formulations according to themethod described in Example 19, above. The static binding capacities forthese samples are provided in Table 3.

The data in Table 3 that the BSA static binding capacities arecomparable for all depth filter media formulations. In addition, as seenin Table 3, the type of silica filter aid had a strong effect on themyoglobin and HCP static binding capacity. The two depth filter mediaformulations prepared using the Sipernat 120 filter aid gave lowermyoglobin and HCP static binding capacity values of around 18 mg/g and 3mg/g for myoglobin and HCP, respectively.

The embodiments of the invention wherein the two formulations preparedusing coarse silica particles resulted in increased myoglobin and HCPstatic binding capacity values of 49 mg/g and 6 mg/g, respectively.These results were surprising as it was not originally expected that theparticular types of silica filter aids used would provide substantiallydifferent adsorption properties or binding capacities within thisparticular application. These results suggest that the type of silicafilter aid employed in the depth filter media formulation stronglyinfluences the performance of the filter media with regard to proteinand impurity binding capacities and the adsorptive media performancecharacteristics in the target application.

TABLE 3 BSA, myoglobin and HCP SBC for selected depth filter mediaformulations. Depth Filter BSA SBC Myoglobin SBC HCP SBC Media ID (mg/g)(mg/g) (mg/g) 2-1 12 16 4 2-2 17 20 2 2-3 16 49 6 2-4 23 49 6

Example 22

Static binding capacity measurements of selected depth filter mediaformulations.

BSA, myoglobin, and HCP static binding capacity measurements wereperformed for various depth filter media formulations according to themethod described in Example 19, above. The static binding capacities forthese samples are provided in Table 4.

The data in Table 4 shows that the BSA, myoglobin, and HCP staticbinding capacity values are not significantly affected by large changesin the total silica filter aid loading or the blend ratio of the twosilica particle sizes (coarse silica particles and fine silicaparticles). The elimination of the wet-strength binder resin in depthfilter media formulations 3-1 thru 3-4 resulted in only a smallreduction in the BSA static binding capacity for these fourformulations.

TABLE 4 BSA, myoglobin and HCP SBC for selected depth filter mediaformulations. Depth Filter BSA SBC Myoglobin SBC HCP SBC Media ID (mg/g)(mg/g) (mg/g) 3-1 6 34 4 3-2 8 44 5 3-3 6 35 4 3-4 12 33 5 3-5 12 37 43-6 12 44 7 3-7 16 33 5 3-8 18 45 8 3-9 14 43 6

Example 23

Static Binding Capacity Measurements of Selected Depth Filter MediaFormulations.

BSA, myoglobin, and HCP static binding capacity measurements wereperformed for various depth filter media formulations according to themethod described in Example 19, above. The static binding capacities forthese samples are provided in Table 5.

The data in Table 5 shows low myoglobin static binding capacity valuesfor all of the comparative depth filter media formulations evaluated.These comparative depth filter media formulations are constructed usingcellulose pulp, diatomaceous earth (DE) filter aid, and the same Wetstrength resin C® wet-strength binder resin. These examples providefurther evidence of the unexpected adsorptive properties of the coarsesilica particle filter aid in contrast to the DE filter aids typicallyemployed in depth filter media formulations.

It was also discovered that the incorporation of Wet strength resin C®wet-strength binder resin in examples 4-5 thru 4-9 resulted in asignificant increase in BSA static binding capacity. This result isconsistent with an adsorptive electrostatic interaction between thenegatively charged BSA and the cationic wet-strength binder resin at theapplication pH of 7.3. The HCP static binding capacity was low for thefour depth filter media formulations evaluated by HCP ELISA assay.

TABLE 5 BSA, myoglobin and HCP SBC for selected depth filter mediaformulations. Depth Filter BSA SBC Myoglobin SBC HCP SBC Media ID (mg/g)(mg/g) (mg/g) comparative −2 2 1 example 4-1 comparative −4 −1 example4-2 comparative −5 4 example 4-3 comparative −8 5 example 4-4comparative 22 0 example 4-5 comparative 13 1 2 example 4-6 comparative12 1 example 4-7 comparative 13 1 1 example 4-8 comparative 17 1 3example 4-9

Example 24

Static Binding Capacity Measurements of Selected Depth Filter MediaFormulations.

BSA, myoglobin, and HCP static binding capacity measurements wereperformed for various depth filter media formulations according to themethod described in Example 19, above. The static binding capacities forthese samples are provided in Table 6.

The data in Table 6 shows low BSA, myoglobin, and HCP static bindingcapacity values for comparative examples of depth filter mediaformulations 6-2 thru 6-4. These depth filter media formulations wereprepared using either PAN only, cellulose only, or a mixture ofcellulose and DE filter aid. In contrast, the incorporation of largesilica particles filter aid into the 6-1 depth filter media formulationaffords a modest BSA SBC and high myoglobin and HCP static bindingcapacity values. These examples provide a further evidence of thespecial adsorptive properties of the EMD silica filter aid in contrastto the DE filter aid and other materials of construction that aretypically employed in such depth filter media formulations.

TABLE 6 BSA, myoglobin and HCP SBC for selected depth filter mediaformulations. Depth Filter BSA SBC Myoglobin SBC HCP SBC Media ID (mg/g)(mg/g) (mg/g) 6-1 4 42 5 6-2 −1 1 1 6-3 0 1 1 6-4 −5 1 0

Example 25

Depth Filter Compositions for Clarification Application Testing.

Depth filtration devices were constructed using the selected depthfilter media compositions and non-woven media as shown in the Table 7.These depth filtration devices were utilized in applications testingdirected to the primary and secondary clarification of mAb producing andnon-producing HCCF feedstreams.

TABLE 7 Depth filter compositions for clarification application testing.Depth Filter Device ID Layer 0 Layer 1 Layer 2 7-1 mixed synthetic fiberdepth filter depth filter non-woven (300 g/m², 4 media ID 1-10 media ID1-9 mm thickness) 7-2 synthetic fiber depth filter depth filternon-woven, 2 layers (34 media ID 2-3 media ID 2-4 g/m², 0.1 mmthickness) 7-3 synthetic fiber depth filter depth filter non-woven, 2layers (34 media ID 2-3 media ID 2-4, g/m², 0.1 mm thickness) pressed

Example 26

Improved Filtration Performance and HCP Impurity Clearance (mAb05 Feed).

FIG. 6 provides the pressure and HCP impurity clearance profiles for adepth filter clarification applications test using a HCCF containing amonoclonal antibody (mAb05) with a viable cell density 1.41×10⁷ vc/mL.

The 23 cm² depth filter devices were constructed using the non-woven anddepth filter media grades described in example 25. The filtration andimpurity clearance performance of these devices were compared tocommercial Millistak+® D0HC and X0PS devices (EMD Millipore, Billerica,Mass.). In these tests, the primary and secondary clarification depthfilters were configured in a 2:1 area ratio.

A 2:1 area ratio is defined herein as two parallel primary filterscoupled to a single secondary filter. In this example, two 7-1 depthfilters and two D0HC devices served as the primary clarificationfilters. The 7-2 depth filter and the X0HC device served as thesecondary clarification filters. The two 7-1 depth filters were coupledto the 7-2 depth filter device and the two D0HC devices were coupled tothe X0HC device.

Un-clarified HCCF was pumped through the devices at a flow rate of 150LMH (versus the primary filters) and 300 LMH (versus the secondaryfilter) and the pressure was monitored continuously by means of a systemof pressure transducers and data logging equipment. The filtrate wasfractionated and submitted for HCP ELISA and PicoGreen® DNA assays. Thepressure profile data presented in FIG. 6 shows the discoveredadvantages for the coupled 7-1/7-2 depth filter devices when compared tothe coupled D0HC/X0HC devices for this HCCF feedstream.

Terminal pressure is reached for the D0HC devices at a throughput of 112L/m², while similar pressures are not reached for the 7-1 devices untila throughput of 162 L/m². Similarly, the 7-2 depth filter device showsmodestly lower pressures than the X0HC device at a much high feedthroughput.

The HCP impurity clearance data also presented in FIG. 6 shows HCP/DNAimpurity breakthrough for both the coupled D0HC/X0HC devices and the ID7-1/ID 7-2 depth filter formats evaluated. This data shows thediscovered advantages for the coupled ID 7-1/ID 7-2 devices compared tothe coupled D0HC/X0HC format, as the D0HC/X0HC devices show complete HCPbreakthrough at a throughput of 100 L/m², while significant HCP impurityclearance for the ID 7-1/ID 7-2 devices is still observed until about200 L/m².

Example 27

HCP and DNA Impurity Clearance (mAb05 Feed).

FIG. 7 provides the pressure, HCP, and DNA impurity clearance profilesfor a depth filter secondary clarification applications test using aHCCF containing a monoclonal antibody (mAb05) with a viable cell density8.38×10⁶ vc/mL.

The 23 cm² depth filter devices were constructed using the non-woven anddepth filter media grades described in example 25. In these tests, asufficient quantity of the HCCF was clarified through the prototypeprimary clarification depth filter device 7-1 described above. Thefiltrate was pooled and subsequently processed through the prototypesecondary clarification depth filter devices 7-2 and 7-3 at a flow rateof 300 LMH (versus each secondary filter) and the pressure was monitoredcontinuously by means of a system of pressure transducers and datalogging equipment.

The filtrate was fractionated and submitted for HCP ELISA and PicoGreen®DNA assays. The pressure profile data presented in FIG. 7 shows acomparable performance for the un-coupled 7-2 and 7-3 prototype depthfilter devices for this HCCF feedstream. The HCP and DNA impurityclearance data also presented in FIG. 7 shows a significant clearance ofboth HCP and DNA impurities for each of the 7-2 and 7-3 prototypedevices evaluated at throughputs as high as 120 L/m². This dataindicates acceptable performance for the 7-2 and 7-3 secondaryclarification devices for HCP and DNA impurity clearance within thetargeted clarification application.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions taught herein.

We claim:
 1. A process for producing depth filters for the clarificationof biological fluids comprising: a. providing producing a syntheticcomposite depth filter media comprising one layer, wherein the layercomprises synthetic fibrillated polymeric fibers comprisingpolyacrylonitrile and/or polyacrylonitrile copolymers, and a syntheticfilter aid comprising one or more of silica, alumina, glass, metaloxides or mixed-metal oxides, ion-exchange resins and carbon, whereinsaid fibers and filter aid are processed by a wet-laid process,air-laying process, a melt-pressing process or mechanical compression;and, b. treating said depth filter media by i) irradiating the depthfilter media at a dose of about 10 to about 45 kGy or, ii) flushing thedepth filter media with 0.5 NaOH for about 100 to about 300 LMH forabout 30 min.
 2. The process of claim 1, wherein the synthetic fibercomprises at least 26% by weight based on the total weight of the fiberand filter aid.
 3. The process of claim 1, wherein the filter aidcomprises over 0 to 90% by weight based on the total weight of thesynthetic fibers and filter aid.
 4. The process of claim 1, wherein thedepth filter further comprises a wet strength resin, said wet strengthresin comprising water-soluble synthetic polymers of urea ormelamine-formaldehyde based polymers, polyaminopolyamide-epichlorohydrin(PAE) polymers or glyoxalated polyacrylamide (GPAM) resins.
 5. Theprocess of claim 4, wherein the content by weight of the wet strengthresin based on the total weight of the synthetic fiber and filter aid isover 0% to about 5%.
 6. The process of claim 1, wherein the compositedepth filter media comprises at least one layer comprising a syntheticnonwoven or a microfiber layer, said nonwoven or synthetic microfibercomprising polypropylene, polyesters, polyethylene, nylon,polyacrylonitrile, carbon or glass.
 7. The process of claim 1, whereinthe feed flow direction is from the first layer to the second layer. 8.The process of claim 1, wherein the first filter layer is in directcontact with the second filter layer.
 9. The process of claim 1, whereinsaid level of total organic extractables (TOC) is 2.0 parts per million(ppm) or less.
 10. The process of claim 1, wherein said fibers andfilter aid are processed by a wet-laid process.
 11. The process of claim1, further comprising c) flushing the synthetic depth filter media withwater at flow rates from about 10 liter/m²/hr to about 600 liter/m²/hrsuch that the level of total organic extractables measured in thefiltrate is about 0 to 3 ppm.